STUDIES OF THE PROCESSING OF THE PROTEASE WHICH INITIATES DEGRADATION OF SMALL, ACID-SOLUBLE PROTEINS DURING GERMINATION OF SPORES OF BACILLUS SPECIES

Three mutant forms of the protease (GPR) that initiates degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus species have been generated. In one variant (GPR(Delta)), the putative pro sequence removed in conversion of the GPR zymogen (termed P-46) to the active enzyme (termed P-41) was deleted. GPR(Delta) was expressed in both Escherichia coli and Bacillus subtilis as a polypeptide of 41 kDa (P-41) which was active both in vivo and in vitro. The other two variants had changes in the sequence around the site where the pro sequence is removed, making this sequence even more like that recognized and cleaved by GPR in its SASP substrates. One of these variants (GPR(S)) was synthesized as P-46(S) in both B. subtilis and E. coli, but P-46(S) was processed to P-41(S) earlier in B. subtilis sporulation than was wild-type P-46. The second variant (GPR(EI)) was made as P-46(EI) but underwent extremely rapid processing to P-41(EI) in both E. coli and B. subtilis. Expression of elevated (>100-fold) levels of GPR(Delta) or GPR(EI) blocked sporulation at the time of synthesis of glucose dehydrogenase. Expression of elevated levels of GPR(S) or low levels (<20% of the wild-type level) of GPR(Delta) or GPR(EI) did not retard sporulation, but the SASP level in the resultant spores was greatly reduced. Prolonged incubation of P-41, P-41(EI), or wild-type P-41, either in vivo or with purified proteins in vitro, resulted in a second self-cleavage event generating a 39-kDa polypeptide termed P-39. The sequence in the P-41-->P-39 cleavage site was also quite similar to that recognized and cleaved by GPR in SASP. Together, these results strongly support a model in which activation of GPR during sporulation by conversion of P-46 to P-41 is a self-processing event triggered by a change in the spore core environment (i.e., dehydration) which precludes attack of the active P-41 on its SASP substrates. However, in the first minutes of spore germination, rapid spore core hydration allows rapid attack of active GPR on SASP.